Downregulation of microRNA-196a enhances the sensitivity of non-small cell lung cancer cells to cisplatin treatment

  • Authors:
    • Qian Li
    • Zailiang Yang
    • Mingyan Chen
    • Ying Liu
  • View Affiliations

  • Published online on: March 1, 2016     https://doi.org/10.3892/ijmm.2016.2513
  • Pages: 1067-1074
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Abstract

MicroRNAs (miRNAs or miRs) are a class of small, non-coding RNA molecules that play an important role in the pathogenesis of human diseases through the regulation of gene expression. Although miRNA-196a has been implicated in the progression of human lung cancer, its role in enhancing the sensitivity of non‑small cell lung cancer (NSCLC) cells to cisplatin has not yet been confirmed. The aim of this study was to evaluate the effects of miRNA‑196a on the sensitivity of NSCLC cells to cisplatin in vitro and in vivo. RT-qPCR was used to detect miRNA-196a expression. Synthesized locked nucleic acid (LNA)-anti‑miRNA-196a oligonucleotide was transiently transfected into the SPC‑A‑1 and A549 lung cancer cells to examine the effects of miRNA‑196a on the growth of and colony formation inthe cisplatin‑treated cells. The effects of miRNA-196a on the sensitivity of SPC‑A-1 cells to cisplatin in vivo were determined using BALB/c nude mice. The expression of miRNA‑196a was significantly higher in both the lung cancer tissues and cell lines. The LNA-based knockdown of miRNA-196a significantly inhibited SPC‑A‑1 and A549 cell growth and induced apoptosis. Moreover, the downregulation of miRNA-196a sensitized the SPC‑A‑1 and A549 NSCLC cells to cisplatin in vitro and in vivo, by inducing apoptosis. The findings of this study demonstrate that the administration of cisplatin in combination with miRNA-196a-targeted therapy may be a potential therapeutic strategy for the treatment of NSCLC.

Introduction

Lung cancer is the leading cause of cancer-related mortality worldwide (1,2), and non-small cell lung cancer (NSCLC) accounts for the majority of all lung cancer-related deaths (3). The prognosis of lung cancer remains unfavorable, with a 5-year overall survival rate of approximately 11%, despite recent advances in clinical and experimental oncology (4). Thus, detailed research into the development and progression of NSCLC is essential for improving the diagnosis, prevention and treatment of this disease. Cisplatin (DDP) remains the most widely used first-line chemotherapeutic agent for the treatment of NSCLC. However, the continuous and multiple-dose administration of DDP often causes severe side-effects and cancer cells often become resistant; thus, this has limited the use of this drug (5). Therefore, enhancing the sensitivity of cancer cells to DDP (perhaps to lower doses of the drug) remains a challenge for the efficacty of chemotherapy. Recently, an increasing number of studies has demonstrated that small, non-coding RNAs may be involved in the pathogenesis of NSCLC (6,7,14), thereby providing new insight into the biology of the disease. Previous studies have indicated that the dysregulation of microRNAs (miRNAs or miRs) contributes to the resistance of human cancer cells to DDP (8,9). Novel therapeutic modalities combining miRNAs have the potential to be effective in the treatment of NSCLC in the future.

miRNA-196a (miR-196a) was one of the first miRNAs to be discovered in human cells; it is highly conserved in mammals. Previous studies have indicated that the expression of miR-196a is significantly upregulated in different solid tumors (1012) and have revealed that miR-196a is involved in the proliferation, detachment, migration and invasion of a number of cancer cells (colorectal cancer, breast cancer, pancreatic cancer, gastric cancer and NSCLC cells) (1216). Huang et al reported that miR-196a promotes the progression of pancreatic cancer (13); Liu et al found that the downregulation of miR-196a inhibited NSCLC cell proliferation and invasion (14). However, to the best of our knowledge, there are no studies available to date on the association between miR-196a expression and the sensitivity of NSCLC cells to DDP.

In the present study, we found that miR-196a was upregulated in human NSCLC tissues and cell lines; the downregulation of miR-196a enhanced the sensitivity of NSCLC cell lines (SPC-A-1, A549) to DDP through the induction of apoptosis by targeting homeobox A5 (HOXA5). Taken together, these findings suggest that miR-196a is a valid therapeutic target with the potential to be employed as a novel multimodality therapy as part of a strategy for the treatment of patients with NSCLC.

Materials and methods

Patients and tissue samples

A total of 23 pairs of matched NSCLC and non-cancerous tissue samples were obtained from patients undergoing surgical procedures at the Sixth People's Hospital of Chongqing (Chongqing, China), and tumor diagnosis was performed by an independent pathologist. None of the patients had received chemotherapy or radiotherapy prior to surgery. The samples were snap-frozen in liquid nitrogen and stored at ‒80°C until RNA extraction. Written informed consent was obtained from all patients prior to surgery. This study was approved by the Research Ethics Committee of the Sixth People's Hospital of Chongqing, China.

Animals

Female BALB/c nude mice (n=48, 4–6 weeks of age) were purchased from the Shanghai Laboratory Animal Research Center (Shanghai, China) and maintained under pathogen-free conditions. All experimental procedures involving animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were performed according to the Institutional Ethical Guidelines for Animal Experiments.

Cell culture

Four NSCLC adenocarcinoma cell lines (A549, SPC-A-1, NCI-H1650 and NCI-H1299), a NSCLC squamous carcinoma cell line (SK-MES-1) and a normal human bronchial epithelial cell line (16HBE) were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (both from Gibco, Grand Island, NY, USA), 100 U/ml penicillin and 100 mg/ml streptomycin (both from Invitrogen, Carlsbad, CA, USA) in humidified air with 5% CO2 at 37°C.

Locked nucleic acid (LNA)-anti-miR-196a oligonucle-otidetransfection assay

The SPC-A-1 and A549 cells were maintained in RPMI-1640 medium. For transfection, LNA-anti-miR-196a or LNA-control oligonucleotides were delivered at a final concentration of 50 nM [as previously described (17)] using Lipofectamine 2000 reagent (Invitrogen). LNA-anti-miR-196a and LNA-control oligonucleotides were purchased from Exiqon A/S (Vedbaek, Denmark). A group of mock cells (untransfected cells) was also used.

Treatment of cells with DDP

The mock SPC-A-1/A549 cells and the SPC-A-1/A549 cells transfected with LNA-anti-miR-196a/LNA-control oligonucleotide were treated with various concentrations (0, 5, 10 and 20 μg/ml) of DDP for 12 h or 5 μg/ml of DDP for 0, 24, 48, 72 and 96 h.

Cell viability assay

Cell viability was assessed using the CCK8 assay (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. Briefly, the treated cells were cultured in triplicate in a 96-well plate. CCK8 reagent was added to each well 2 h prior to the termination of the experiment, and the absorbance (OD450) was expressed as the viability percentages of the cells compared with the controls. All tests were performed in triplicate and the data are presented as the means ± SD.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the mock-transfected SPC-A-1/A549 or stably-transfected SPC-A-1/A549 cells (5×106 cells) and the tissue samples, using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. To quantify the miRNA levels, RT-qPCR was performed using TaqMan microRNA assays (Applied Biosystems Life Technologies, Foster City, CA, USA). Briefly, 10 ng of total RNA was reverse transcribed using an miRNA-specific looped RT primer for each miRNA and a corresponding TaqMan® microRNA Reverse Transcription kit (Applied Biosystems Life Technologies; prime sequences included in kit). qPCR was performed using the generated cDNA in gene-specific TaqMan miRNA Real-Time PCR assay solution on a StepOnePlus Real-Time PCR system (Applied Biosystems Life Technologies). The reaction was performed at 95°C for 10 min, followed by 45 cycles at 95°C for 15 sec, and 60°C for 60 sec. RNA U6 (RNU6B; Applied Biosystems Life Technologies) was used as an internal control. The relative expression was calculated using the comparative cycle threshold (Ct) method. All qPCR reactions were performed in triplicate and the data are presented as the means ± SD.

Colony formation assay

Approximately 500 mock-transfected SPC-A-1/A549 or stably-transfected SPC-A-1/A549 cells were placed in a fresh 6-well plate with or without DDP for 12 h and maintained in RPMI-1640 containing 10% FBS for another 2 weeks. The colonies were fixed with methanol and stained with 0.1% crystal violet in 20% methanol for 15 min. All samples were analyzed in triplicate and the data are presented as the means ± SD.

Flow cytometric analysis of apoptosis

The mock-transfected SPC-A-1/A549 or stably-transfected SPC-A-1/A549 cells were treated with DDP and harvested. The cells were double-stained with FITC-Annexin V and propidium iodide (PI), and then analyzed using a flow cytometer equipped with CellQuest software (both from BD Biosciences, Franklin Lakes, NJ, USA), as previously described (18). The relative ratio of apoptotic cells was compared with the control from each experiment. All samples were analyzed in triplicate and the data are presented as the means ± SD.

Caspase-3 activity assay

Briefly, the cells were seeded in a 6-well plate, cultured for 24 h and then treated with or without DDP for a further 12 h, and harvested. The activity of caspase-3 was measured using a Caspase-3 Colorimetric assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Caspase-3 activity was quantified spectrophotometrically at a wavelength of 405 nm. All samples were analyzed in triplicate and the data are presented as the means ± SD.

Western blot analysis

Protein extracts (40 μg) from the treated cells were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Invitrogen) and electrophoretically transferred onto PDVF membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membranes were blocked with 5% non-fat dried milk for 2 h, and then incubated for 2 h with specific primary antibodies HOXA5 (sc-365784), p53 (sc-126) and β-actin (sc-376421) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween-20), the membranes were incubated with horseradish peroxidase-linked antibody (#7076; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h. The membranes were washed and the proteins were visualized using ECL chemiluminescence and exposed to X-ray film. All of the samples were analyzed in triplicate.

In vivo experiments using mice

BALB/c nude mice were randomly divided into 6 groups [mock + vehicle (normal saline), mock + DDP, LNA-NC + vehicle, LNA-NC + DDP, LNA-anti-miR196a + vehicle and LNA-anti-miR196a + DDP; 8 mice/group]. The mice were injected with either mock-transfected or stably-transfected SPC-A-1 cells by subcutaneous injection (3×106 cells/0.2 ml). One day after tumor cell implantation, the mice were treated with DDP [3.0 mg/kg; intraperitoneally (i.p.) every other day (qod)] or the vehicle. Tumor volume was examined for 5 weeks and measured once a week. The volume formed was calculated using the following formula: volume = 0.4 × D × d2 (D, longitudinal diameter; d, latitudinal diameter). All mice were sacrificed by exposure to carbon dioxide and the tumors were harvested, and TUNEL staining assay was then performed.

TUNEL staining assay

The tissues were harvested and plated on polylysine-coated slides. The slides were then fixed with 4% paraformaldehyde for 1 h at room temperature, and were then rinsed with 0.1 M PBS, and finally permeabilized with 1% Triton X-100. DNA fragmentation was detected using the TUNEL Apoptosis Detection kit (KeyGen, Nanjing, China), which specifically labeled 3′-hydroxyl termini of DNA strand breaks using fluorescein isothiocyanate (FITC)-conjugated dUTP. The DNA was labeled with FITC DNA-binding dye for 5 min. The FITC fluorescence was measured using a fluorescence microscope. The percentage of apoptotic cells was calculated as the number of apoptotic cells per the number of total cells ×100.

Statistical analysis

In the present study, the data are expressed as the means ± SD. Statistical analysis was performed using a t-test with SPSS 13.0 software to evaluate the significance of differences between groups. A p-value <0.05 was considered to indicate a statistically significant difference.

Results

miR-196a expression is upregulated in human NSCLC tissues and cell lines

In this study, the miR-196a levels were measured in 23 NSCLC samples and adjacent normal tissues by RT-qPCR, and normalized to U6. The results indicated that miR-196a expression was significantly upregulated in the NSCLC samples compared with the expression levels in the corresponding normal tissue samples (Fig. 1A). We also performed RT-qPCR to examine the expression of miR-196a in the human NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes. The results revealed that miR-196a expression was also significantly upregulated in the human NSCLC cell lines compared with the expression levels in the normal cell line, 16HBE (Fig. 1B). These results indicate that the overexpression of miR-196a may play an important role in the progression and development of NSCLC.

Manipulation of miR-196a levels in NSCLC cells by performing LNA-anti-miR-196a oligonucleotide transfection assay

To selectively downregulate miR-196a in the NSCLC cell lines, SPC-A-1/A549, the LNA-anti-miR-196a oligonucleotide transfection assay was used in this study. The SPC-A-1/A549 cells were transfected with LNA-anti-miR-196a or LNA-control oligonucleotide; 48 h after transfection, the cells were harvested and RT-qPCR was performed. The results revealed that the expression of miR-196a was significantly downregulated by approximately 4-5-fold following transfection with LNA-anti-miR-196a oligonucleotide compared with that in the mock-transfected cells (Fig. 2); therefore, transfection with LNA-anti-miR-196a oligonucleotide was used to manipulate the miR-196a level to investigate the biological effects of miR-196a in the subsequent experiments.

Effect of miR-196a on NSCLC cell proliferation and apoptosis

To examine the biological role of miR-196a in NSCLC cells, we examined the effects of downregulating miR-196a on cell proliferation and apoptosis by CCK8 assay, colony formation assay and FACS analysis. As shown in Fig. 3A and B, the SPC-A-1 and A549 cells transfected with LNA-anti-miR-196a oligonucleotide exhibited a significant decrease in cell viability compared with the mock- or LNA-control-transfected cells, particularly after 96 h (p<0.001). Similarly, the results of colony formation assay revealed that clonogenic survival was decreased following the downregulation of miR-196a in the SPC-A-1 and A549 cells compared with the the mock- or LNA-control-transfected cells (p<0.001; Fig. 3C). To determine whether apoptosis was a contributing factor to cell growth inhibition, we performed flow cytometric analysis of the SPC-A-1 and A549 cells following transfection with LNA-anti-miR-196a oligonucleotide. The results revealed that the apoptotic rate was significantly increased in the SPC-A-1/A549 cells transfected with LNA-anti-miR-196a oligonucleotide compared with that in the mock- or LNA-control-transfected cells (p<0.01) (Fig. 3D). Taken together, these results indicate that the inhibition of miR-196a may inhibit the growth and induce the apoptosis of SPC-A-1 and A549 cells.

Downregulation of miR-196a enhances the sensitivity of NSCLC cells to DDP in vitro

The dysregulation of miRNA expression has been reported to be associated with chemoresistance in human cancers (19). However, whether miR-196a expression affects the sensitivity of NSCLC cells to DDP is not yet fully understood. In the present study, we hypothesized that there was an association between the dysregulation of miR-196a and the sensitivity of NSCLC cells to DDP. To examine this hypothesis, the mock-transfected SPC-A-1/A549 cells and the SPC-A-1/A549 cells transfected with LNA-anti-miR-196a/LNA-control oligonucleotide were treated with various concentrations (0, 5, 10 and 20 μg/ml) of DDP for 12 h or 5 μg/ml of DDP for 0, 24, 48, 72 and 96 h. The CCK8 assay was performed to determine cell viability. The results indicated that the downregulation of miR-196a led to a significant decrease in the viability of the SPC-A-1/A549 cells treated with DDP, in a dose- and time-dependent manner compared with that in the LNA-control and the mock-transfected cells (p<0.001; Fig. 4). These data clearly demonstrate that the downregulation of miR-196a may effectively enhance the sensitivity of NSCLC cells to DDP.

Downregulation of miR-196a enhances the DDP-induced apoptosis of NSCLC cells

To determine whether apoptosis was a contributing factor to the enhanced sensitivity of SPC-A-1/A549 cells to DDP, we performed flow cytometric analysis. As shown in Fig. 5A, the apoptotic rate was significantly increased in the SPC-A-1/A549 cells transfected with LNA-anti-miR-196a oligonucleotide and treated with 5 μg/ml DDP compared with that in the mock-transfected cells treated with 5 μg/ml DDP. The apoptotic rate of the SPC-A-1/A549 cells transfected with LNA-control oligonucleotide and treated with DDP did not differ significantly compared with that of the mock-transfected cells treated with DDP. We then examined caspase-3 activity using a colorimetric assay. The results revealed that caspase-3 activity in the SPC-A-1/A549 cells transfected with LNA-anti-miR-196a oligonucleotide and treated with DDP significantly increased compared with that in the mock- or LNA-control-transfected cells treated with DDP (Fig. 5B). Therefore, the downregulation of miR-196a may increase the sensitivity of SPC-A-1/A549 cells to DDP by enhancing DDP-induced apoptosis.

Downregulation of miR-196a enhances HOXA5 expression in NSCLC cells

A previous study indicated that miR-196a promotes NSCLC cell proliferation and invasion by targeting HOXA5 (14). Raman et al reported that the overexpression of HOXA5 induced cell apoptosis and the overexpression of p53 concomitantly (20). Thus, to determine whether the apoptosis induced by the downregulation of miR-196a was affected by HOXA5, we performed western blot analysis to determine the HOXA5 and p53 levels in the cisplatin-treated NSCLC cells. The results revealed that the downregulation of miR-196a enhanced the expression of HOXA5 and p53 in the SPC-A-1/A549 cells treated with 5 μg/ml DDP (Fig. 6). RT-qPCR was then performed to determine the mRNA levels of HOXA5 and p53, and the results of RT-qPCR were similar to those of western blot analysis (data not shown). Therefore, the downregulation of miR-196a induced the expression of HOXA5 and p53 at the protein and mRNA level. Taken together, these findings suggest that the downregulation of miR-196a enhances the sensitivity of NSCLC cells to DDP through apoptotic signaling by targeting HOXA5. We aim to elucidate the precise mechanism of this interaction with apoptotic signaling in future studies.

Downregulation of miR-196a enhyances the sensitivity of SPC-A-1 cells to DDP in vivo

To examine the effects of downregulating miR-196a on the sensitivity of SPC-A-1 cells to DDP in vivo, nude mice were subcutaneously injected with SPC-A-1 cells to form tumors. This was followed by the administration of either DDP or the vehicle. The results revealed that the tumors formed following transfection with LNA-anti-miR-196a oligonucleotide grew significantly slower than those from the mice injected with the mock- or LNA-control-transfected SPC-A-1 cells (Fig. 7A). Following treatment with DDP, the inhibition of tumor growth in the mice injected with the SPC-A-1 cells transfected with LNA-anti-miR-196a oligonucleotide was much greater than that in the mice injected with mock- or LNA-control-transfected SPC-A-1 cells (p<0.05). The results of TUNEL assay revealed that the apoptotic rate in the tumors of mice injected with the SPC-A-1 cells transfected with LNA-anti-miR-196a oligonucleotide was significantly higher than that of the tumors from the mice injected with the mock- or LNA-control-transfected SPC-A-1 cells following treatment with DDP (p<0.001; Fig. 7B). These data clearly indicate that the downregulation of miR-196a may effectively enhance the sensitivity of SPC-A-1 cells to DDP by inducing apoptosis in vivo.

Discussion

It is well known that miRNAs are a series of small (19–24 nt in length), non-coding RNAs which are involved in post-transcriptional gene regulation or degradation (21,22). In a wide range of plant and animal cells, miRNAs have been shown to play an important role in various processes, including cell proliferation, differentiation and metabolism (23,24). They bind to target mRNAs at the 3′-untranslated region (UTR) and/or 5′-UTR of target mRNA, to block translation or contribute to target mRNA degradation (25). There is increasing evidence to suggest that the deregulation of miRNAs frequently occurs in tumor tissues, and that they target genes involved in cancer cell proliferation, differentiation, apoptosis, metastasis and resistance (2629). It has also been demonstrated that miRNAs play an important role in modulating sensitivity and resistance to anticancer drugs in substantial ways (19).

Recently, the function of miR-196a in the pathogenesis of tumors has been widely investigated; a number of studies have suggested that miR-196a exhibits an oncogenic function in cancer (3033,40). Higher levels of miR-196a have been found in pancreatic cancer, leukemia and esophageal adenocarcinoma, and have been shown to be negatively associated with survival (3438). In esophageal cancer, miR-196a overexpression has been shown to promote cell proliferation and to suppress apoptosis by directly regulating annexin A1 (39). In colorectal cancer, high levels of miR-196a have been shown to promote cancer cell detachment, migration, invasion and chemosensitivity, and to promote the development of lung metastases in mice by activating the Akt signaling pathway (11,40). However, the mechanisms responsible for the chemosensitivity mediated by miR196a have not yet been clearly defined and evidence of an association between miR-196a and chemoresistance remains limited. In addition, the potential involvement of miR-196a in the induction of drug resistance, particularly in resistance to DDP, remains to be determined. Inspired by the above-mentioned observations, in this study, we investigated the biological role of miR-196a in mediating resistance to DDP in NSCLC cells.

In this study, we demonstrated that miR-196a expression was upregulated in NSCLC tissue samples and NSCLC cell lines. In addition, we attempted to explore the role of miR-196a in NSCLC; the results revealed that the targeted knockdown of miR-196a expression in NSCLC cells led to the significant inhibition of cell proliferation and colony formation by inducing caspase-3-dependent apoptosis. Furthermore, we found that the downregulation of miR-196a significantly enhanced the sensitivity of NSCLC cells to DDP in vitro and in vivo, by targeting HOXA5. However, further studies exploring the role of miR-196a in the regulation of NSCLC cell growth and metastasis are warranted in the future.

miR-196a plays critical roles in the pathogenesis of cancer by targeting several genes, including high mobility group AT-hook 2 (HMGA2), annexin A1 and HOX; however, miR-196a is not only expressed in cancer cells, but also in normal cells (4043), and the functions and targets of miR-196a have not yet been fully analyzed. The downregulation of miR-196a may induce abnormal gene expression in normal cells. As a potential concern, the miRNA antagonist used may also non-specifically bind to other RNAs, and one miRNA may have multiple target genes due to the sequence homology of the binding sites, which may result in unwanted side-effects. It has been reported that single-nucleotide polymorphism (SNP) in miRNA-196a is associated with severe toxicity in lung cancer patients, particularly in individuals treated with cisplatin or gemcitabine (44). In spite of the multiple potential side-effects, miR-196a gene therapy may prove to be a novel multimodality treatment for NSCLC with continued research.

Taken together, the findings of the present suggest that the downregulation of miR-196a enhances the sensitivity of NSCLC cells to DDP both in vitro and in vivo. Thus, the appropriate application of DDP in combination with the regulation of miR-196a expression may prove to be a potential therapeutic strategy for the treatment of NSCLC in the future.

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April-2016
Volume 37 Issue 4

Print ISSN: 1107-3756
Online ISSN:1791-244X

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Spandidos Publications style
Li Q, Yang Z, Chen M and Liu Y: Downregulation of microRNA-196a enhances the sensitivity of non-small cell lung cancer cells to cisplatin treatment. Int J Mol Med 37: 1067-1074, 2016
APA
Li, Q., Yang, Z., Chen, M., & Liu, Y. (2016). Downregulation of microRNA-196a enhances the sensitivity of non-small cell lung cancer cells to cisplatin treatment. International Journal of Molecular Medicine, 37, 1067-1074. https://doi.org/10.3892/ijmm.2016.2513
MLA
Li, Q., Yang, Z., Chen, M., Liu, Y."Downregulation of microRNA-196a enhances the sensitivity of non-small cell lung cancer cells to cisplatin treatment". International Journal of Molecular Medicine 37.4 (2016): 1067-1074.
Chicago
Li, Q., Yang, Z., Chen, M., Liu, Y."Downregulation of microRNA-196a enhances the sensitivity of non-small cell lung cancer cells to cisplatin treatment". International Journal of Molecular Medicine 37, no. 4 (2016): 1067-1074. https://doi.org/10.3892/ijmm.2016.2513